Stereotypies in Laboratory Mice — Quantitative and Qualitative Description of the Ontogeny of ‘Wire-gnawing’ and ‘Jumping’ in Zur:ICR and Zur:ICR nu

The ontogeny of two stereotypic patterns, wire-gnawing and jumping, was studied in 24 laboratory mice: six males and six females each of two closely related outbred strains, kept under standard housing conditions, a conventional albino strain (ICR) and a nude, athymic mutant (ICR nu; hereafter: NU). All 24 individuals developed wire-gnawing after weaning at 20 d of age. In ICR one female and in NU five males and three females additionally developed jumping. ICR developed wire-gnawing between the age of 20 and 30 d, in NU jumping started at the age of 20 d, but intense jumping and wire-gnawing comparable to that of ICR did not develop in NU before the age of 40–50 d. Within each strain there was no significant difference between males and females with respect to the development of stereotypic behaviour. By contrast, ICR showed significantly more wire-gnawing but less jumping than NU. Stereotypy level increased with age up to a mean of 10.7 % of total activity in ICR and up to 7.4 % in NU at 100 d of age. However, there was huge inter- and intra-individual variability with respect to all parameters assessed in this study, i.e. total duration, number of bouts and bout length of the two stereotyped patterns. Wire-gnawing developed from outside-directed explorative climbing at the cage lid, whereas the source behaviour pattern (Mason 1991 a, Anim. Behav. 41, 1015–1037) of jumping was outside-directed explorative rearing at the cage wall. At 20 d of age, before the onset of stereotypy development, ICR showed significantly more climbing but less rearing than NU. Physical retardation of NU at weaning may account for decreased climbing ability during early ontogeny, and hence for the retarded development of wire-gnawing. The difference in early experience with either of the two patterns rather than genetic effects may be responsible for the qualitative difference between the strains with respect to the form of later stereotypy.     

Lifestyle-related biomarkers and endometrial cancer survival: Elevated gamma-glutamyltransferase as an important risk factor

Background: Lifestyle seems to play an important role in endometrial cancer mortality, but it remains unclear which biomarkers are involved. The aim of this study was to assess the extent of the association between lifestyle-related biomarkers and the survival of endometrial cancer patients. Methods: A sub-cohort of 242 endometrial cancer patients, from a population-based study of the more than 90,000 female participants of the Vorarlberg Health Monitoring and Promotion Programme, was followed for a median duration of twelve years. Besides age, tumour staging, and histology, also pre-diagnostic levels of body mass index, blood pressure, triglycerides, total cholesterol, glucose, gamma-glutamyltransferase (GGT), and serum uric acid were analysed in Cox proportional hazards regression models to estimate multivariate mortality risks. Results: During follow-up 89 deaths occurred of which 49 were cancer-related. Survival was associated with age, tumour stage, and histology. Of the biomarkers, log₁₀-transformed GGT showed a large effect on cancer-related mortality (HR = 3.35, 95% CI 1.12–10.03), whereas the other parameters did not appear with significant effects after adjustment for the other factors. Conclusion: Elevated level of GGT, a lifestyle-related marker, was associated with poor survival among endometrial cancer patients.     

phenoSeeder - A Robot System for Automated Handling and Phenotyping of Individual Seeds

The enormous diversity of seed traits is an intriguing feature and critical for the overwhelming success of higher plants. In particular, seed mass is generally regarded to be key for seedling development but is mostly approximated by using scanning methods delivering only two-dimensional data, often termed seed size. However, three-dimensional traits, such as the volume or mass of single seeds, are very rarely determined in routine measurements. Here, we introduce a device named phenoSeeder, which enables the handling and phenotyping of individual seeds of very different sizes. The system consists of a pick-and-place robot and a modular setup of sensors that can be versatilely extended. Basic biometric traits detected for individual seeds are two-dimensional data from projections, three-dimensional data from volumetric measures, and mass, from which seed density is also calculated. Each seed is tracked by an identifier and, after phenotyping, can be planted, sorted, or individually stored for further evaluation or processing (e.g. in routine seed-to-plant tracking pipelines). By investigating seeds of Arabidopsis (Arabidopsis thaliana), rapeseed (Brassica napus), and barley (Hordeum vulgare), we observed that, even for apparently round-shaped seeds of rapeseed, correlations between the projected area and the mass of seeds were much weaker than between volume and mass. This indicates that simple projections may not deliver good proxies for seed mass. Although throughput is limited, we expect that automated seed phenotyping on a single-seed basis can contribute valuable information for applications in a wide range of wild or crop species, including seed classification, seed sorting, and assessment of seed quality.Seeds play a major role in keeping continuity between successive generations (Esau, 1977) and are key for the distribution and evolution (Moles et al., 2005) of higher plants. Fertile seeds carry an embryo and may contain nutrient storage tissues in cotyledons, endosperm, and/or perisperm, supporting germination and seedling development at early developmental stages. Although this is true for all seed plants, various traits of seeds, such as size, shape, weight, and chemical composition, can be very different between plant species or accessions. For example, the Arabidopsis (Arabidopsis thaliana) accession Cape Verde Islands was reported to yield on average 40% fewer seeds than Landsberg erecta, but they are almost twice as heavy (Alonso-Blanco et al., 1999). Considering today’s plant species, single-seed mass may vary over a range of 11.5 orders of magnitude (Moles et al., 2005). Seed mass is under strong genetic control, whereas the total number of seeds of a plant is largely affected by the environment (Paul-Victor and Turnbull, 2009). It has been demonstrated that the size, mass, and shape of Arabidopsis seeds may be regulated by brassinosteroid (Jiang et al., 2013), and it was shown recently that seed size in rice (Oryza sativa) can be influenced by the epiallele Epi-rav6 (Zhang et al., 2015). The ability of plants to switch between small and larger seeds may be understood as an adaptation to novel environments (Igea et al., 2016). However, it is still not fully understood whether, or to what extent, the variability of seed traits within plant species or genotypes has an impact on the development and further performance of a plant.When comparing biometric seed data of different dimensions such as length (one-dimensional), projected area (two-dimensional [2D]), or volume and mass (both three-dimensional [3D]), one can argue that mass is the most relevant parameter as a proxy for the amount of reserves a seed provides for the offspring. This might be true even when considering that the type of reserves, such as proteins, carbohydrates, or lipids (Rolletschek et al., 2015), and also different seed tissues, such as seed coat, embryo, or endosperm, may contribute differently to seed mass (Alonso-Blanco et al., 1999). While seed mass and time to germination (radicle protrusion) do not necessarily correlate (Norden et al., 2009), in particular under greenhouse conditions, higher seed mass may be advantageous for seedling establishment under adverse environmental conditions (Moles et al., 2005). For example, shade-tolerant species showed largely higher seed masses than cogeneric species growing in open habitats, indicating that seedlings under low-light conditions need more reserves than under good light (Salisbury, 1974). Seedlings of wild radish (Raphanus raphanistrum) emerged more likely from heavier seeds than from small seeds under field conditions but not in the greenhouse (Stanton, 1984), and for Arabidopsis, seed mass was reported to be higher in populations growing naturally at higher altitudes taken as a proxy for harsher conditions (Montesinos-Navarro et al., 2011).Seed mass can be measured individually (Stanton, 1984), but it is generally collected as an average value of batches of 50 to 1,000 seeds (Jako et al., 2001; Jofuku et al., 2005; Montesinos-Navarro et al., 2011; Tanabata et al., 2012). Alternatively, 2D scans are analyzed to determine parameters such as seed length, width, area, and perimeter length as a measure for seed size (Tanabata et al., 2012). This approach can be implemented in high-throughput facilities to obtain projected areas of seed grains combined with genome-wide association studies (Yang et al., 2014). Although projected seed area can easily be measured with a common office scanner (Herridge et al., 2011; Tanabata et al., 2012; Moore et al., 2013), it is not necessarily a precise or reliable measure of the true seed size because it may depend on the shape (Alonso-Blanco et al., 1999) and the orientation of a seed at scan (see “Results”). These issues also apply when using 2D projections to calculate length-to-width ratios as a simple shape factor (Tanabata et al., 2012). Projected seed area also has been used to calculate seed mass, assuming a fixed relationship between these parameters (de Jong et al., 2011; Herridge et al., 2011). This may hold with sufficient accuracy when averaging a large number of seeds but might be misleading when considering individual seeds.From a physical point of view, volume should be a much better proxy for mass than 2D traits. Although it has been stated that for 65 species analyzed seed masses can be compared easily with seed volumes (Moles et al., 2005), it is not clear how these seed volumes were determined. Volumes can be assessed using advanced methods such as x-ray computed tomography (CT) on fruits (Stuppy et al., 2003) or synchrotron radiation x-ray tomographic microscopy applied in paleobiological studies (e.g. on fruits and seed; Friis et al., 2014). Nuclear magnetic resonance (NMR) methods are used to measure water uptake in kidney beans (Phaseolus vulgaris) and adzuki beans (Vigna angularis; Kikuchi et al., 2006) or to estimate seed weight and content (Borisjuk et al., 2011; Rolletschek et al., 2015) rather than volumes. To our best knowledge, affordable methods to measure seed volumes directly are not achievable so far. For that reason, we have set up a volume-carving method for 3D seed shape reconstruction that is described briefly here and in more detail in a recent publication (Roussel et al., 2016).While traits derived from scanning procedures can easily be assigned to individual seeds (Herridge et al., 2011), further handling and processing of phenotyped single seeds is not as simple, in particular for tiny ones like those of Arabidopsis. The aim of this work was to develop an automated seed-handling system that can analyze single seeds of very different sizes or shapes, from Arabidopsis seeds up to barley (Hordeum vulgare) seeds or even bigger. The phenoSeeder system is designed to pick and place seeds, to achieve basic morphometric traits (one-dimensional and 2D data from projections, 3D reconstruction data, and mass) of each individual seed, and to store all analyzed seed traits in a database. Another goal is to use phenoSeeder for seed-to-plant tracking approaches and to analyze whether, or which, particular seed traits have an impact on plant development and performance under various environmental conditions. We describe the main features of the phenoSeeder technology and present results obtained with seeds of three accessions of Arabidopsis, rapeseed (Brassica napus), and barley, respectively. When analyzing the data, we focused particularly on correlations between projected seed area, seed volume, and seed mass, with the hypothesis that the respective seed volume may better correlate with mass than the projected area.     

Taxonomy of the glycerol fermenting clostridia and description of Clostridium diolis sp. nov

Six Clostridium strains which ferment glycerol to 1,3-propanediol were tested for their taxonomic and phylogenetic relatedness. All but one were known as C butyricum. By physiological tests, 16S rDNA sequences and fatty acid composition two groups were distinguished. The first comprised the strains VPI 3266, DSM 2478 and DSM 523 (C. "kainantoi") and was consistent with the type strain of C. butyricum in almost all characters. The second group comprising the strains DSM 5430, DSM 5431 and E5 was related to C. beijerinckii. The 16S rDNAs of these strains were almost identical with that of the type strain of C. beijerinckii, DSM 791. The DNA-DNA hybridization value of DSM 5431 and ES with C. beijerinckii DSM 791 was markedly but not decisively lower (67 and 72%, respectively). However, there were significant physiological differences to C. beijerinckii which suggested to describe the strains as a separate species, Clostridium diolis with strain SH1 (= DSM 5431) as the type strain. The new species is distinguished from C. beijerinckii, which requires complex nutrients, by its ability to grow in glucose mineral medium with biotin as the only growth factor and by differences in substrate utilization. "C. kainantoi" Takeda and Matsui was recognized as a later synonym of C. butyricum.     

Lack of electricity production by Pelobacter carbinolicus indicates that the capacity for Fe(III) oxide reduction does not necessarily confer electron transfer ability to fuel cell anodes

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.     

Deep Genetic Divergence between Disjunct Refugia in the Arctic-Alpine King’s Crown,Rhodiola integrifolia (Crassulaceae)

Despite the strength of climatic variability at high latitudes and upper elevations, we still do not fully understand how plants in North America that are distributed between Arctic and alpine areas responded to the environmental changes of the Quaternary. To address this question, we set out to resolve the evolutionary history of the King’s Crown, Rhodiola integrifolia using multi-locus population genetic and phylogenetic analyses in combination with ecological niche modeling. Our population genetic analyses of multiple anonymous nuclear loci revealed two major clades within R. integrifolia that diverged from each other ~ 700 kya: one occurring in Beringia to the north (including members of subspecies leedyi and part of subspecies integrifolia), and the other restricted to the Southern Rocky Mountain refugium in the south (including individuals of subspecies neomexicana and part of subspecies integrifolia). Ecological niche models corroborate our hypothesized locations of refugial areas inferred from our phylogeographic analyses and revealed some environmental differences between the regions inhabited by its two subclades. Our study underscores the role of geographic isolation in promoting genetic divergence and the evolution of endemic subspecies in R. integrifolia. Furthermore, our phylogenetic analyses of the plastid spacer region trnL-F demonstrate that among the native North American species, R. integrifolia and R. rhodantha are more closely related to one another than either is to R. rosea. An understanding of these historic processes lies at the heart of making informed management decisions regarding this and other Arctic-alpine species of concern in this increasingly threatened biome.     

Familial longevity is marked by enhanced insulin sensitivity

Insulin resistance is a risk factor for various age-related diseases. In the Leiden Longevity study, we recruited long-lived siblings and their offspring. Previously, we showed that, compared to controls, the offspring of long-lived siblings had a better glucose tolerance. Here, we compared groups of offspring from long-lived siblings and controls for the relation between insulin and glucose in nonfasted serum (n = 1848 subjects) and for quantitation of insulin action using a two-step hyperinsulinemic-euglycemic clamp (n = 24 subjects). Groups of offspring and controls were similar with regard to sex distribution, age, and body mass index. We observed a positive bi-phasic linear relationship between ln (insulin) levels and nonfasted glucose with a steeper slope from 10.7mU L(-1) insulin onwards in controls compared to offspring (P = 0.02). During the clamp study, higher glucose infusion rate was required to maintain euglycemia during high-dose insulin infusion (P = 0.036) in offspring, reflecting higher whole-body insulin sensitivity. After adjustment for sex, age, and fat mass, the insulin-mediated glucose disposal rate (GDR) was higher in offspring than controls (42.5 ± 2.7 vs. 33.2 ± 2.7 micromol kg(-1) min(-1) , mean ± SE, P = 0.025). The insulin-mediated suppression of endogenous glucose production and lipolysis did not differ between groups (all P > 0.05). Furthermore, GDR was significantly correlated with the mean age of death of the parents. In conclusion, offspring from long-lived siblings are marked by enhanced peripheral glucose disposal. Future research will focus on identifying the underlying biomolecular mechanisms, with the aim to promote health in old age.